Several members of the protein kinase family have been clearly implicated in the pathogenesis of various proliferative diseases and thus represent important targets for treatment of these diseases. Some of the proliferative diseases relevant to this invention include cancer, rheumatoid arthritis, atherosclerosis, and retinopathies. Important examples of kinases which have been shown to cause or contribute to the pathogensis of these diseases include C-Abl kinase and the oncogenic fusion protein BCR-Abl kinase; PDGF receptor kinase; VEGF receptor kinases; MAP kinase p38α; and the RAF kinase family.
C-Abl kinase is an important non-receptor tyrosine kinase involved in cell signal transduction. This ubiquitously expressed kinase—upon activation by upstream signaling factors including growth factors, oxidative stress, integrin stimulation, and ionizing radiation—localizes to the cell plasma membrane, the cell nucleus, and other cellular compartments including the actin cytoskeleton (Van Etten, Trends Cell Biol. (1999) 9: 179). There are two normal isoforms of Abl kinase: Abl-1A and Abl-1B. The N-terminal half of c-Abl kinase is important for autoinhibition of the kinase domain catalytic activity (Pluk et al, Cell (2002) 108: 247). Details of the mechanistic aspects of this autoinhibition have recently been disclosed (Nagar et al, Cell (2003) 112: 859). The N-terminal myristolyl amino acid residue of Abl-1B has been shown to intramolecularly occupy a hydrophobic pocket formed from alpha-helices in the C-lobe of the kinase domain. Such intramolecular binding induces a novel binding area for intramolecular docking of the SH2 domain and the SH3 domain onto the kinase domain, thereby distorting and inhibiting the catalytic activity of the kinase. Thus, an intricate intramolecular negative regulation of the kinase activity is brought about by these N-terminal regions of c-Abl kinase. An aberrant dysregulated form of c-Abl is formed from a chromosomal translocation event, referred to as the Philadelphia chromosome (P. C. Nowell et al, Science (1960) 132: 1497; J. D. Rowley, Nature (1973) 243: 290). This abnormal chromosomal translocation leads aberrant gene fusion between the Abl kinase gene and the breakpoint cluster region (BCR) gene, thus encoding an aberrant protein called Bcr-Abl (G. Q. Daley et al, Science (1990) 247: 824; M. L. Gishizky et al, Proc. Natl. Acad. Sci. USA (1993) 90: 3755; S. Li et al, J. Exp. Med. (1999) 189: 1399). the Bcr-Abl fusion protein does not include the regulatory myristolylation site (B. Nagar et al, Cell (2003) 112: 859) and as a result functions as an oncoprotein which causes chronic myeloid leukemia (CML). CML is a malignancy of pluripotent hematopoietic stem cells. The p210 form of Bcr-Abl is seen in 95% of patients with CML, and in 20% of patients with acute lymphocytic leukemia. A p185 form has also been disclosed and has been linked to being causative of up to 10% of patients with acute lymphocytic leukemia.
Growth factor receptor kinases contribute to the growth and metastasis of tumors by stimulating the proliferation of endothelial cells, fibroblasts, smooth muscle cells, and matrix proteins. Conditions such as hypoxia can induce tumor cells to secrete growth factors which subsequently result in the growth of new blood vessels to support the tumor. These growth factors include platelet derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta), which subsequently stimulate secretion of other growth factors including vascular endothelial growth factor (VEGF), fibroblast growth factor, and epidermal growth factor (EGF). The formation of new blood vessels, which is known as angiogenesis, also provides the tumor with a route to metastasize to remote secondary sites. Inhibiting angiogenic factors that support stromal growth has been proposed as a useful therapy for treating cancers (R. M. Shaheen et al, Cancer Research (1999) 59: 5412; R. M. Shaheen et al, Cancer Research (2001) 61: 1464). Mutations of the PGDF receptor have also been identified which constitutively active in absence of growth factor. VEGF can also stimulate the formation of new lymphatic vessels through direct action on the so-called VEGF-3 receptor, providing yet another pathway for tumor metastasis. Among the three known VEGF receptors, in particular the so-called VEGFR2 (otherwise known as the kinase insert domain-containing receptor tyrosine kinase or KDR) has been demonstrated to be responsible for the role of VEGF in tumor angiogenesis.
A major signaling pathway downstream of cell surface growth factor receptor activation is the Ras-RAF-MEK-ERK-MAP kinase pathway (Peyssonnaux, C. et al, Biol. Cell (2001) 93: 53-62, Cancers arise when mutations occur in one or more of the proteins involved in this signaling cascade. Cell proliferation and differentiation become dysregulated and cell survival mechanisms are activated which allow unregulated cancer cells to override protective programmed cell death surveillance. Mutations in the p21-Ras protein have been shown to be a major cause of dysregulation of this signaling pathway, leading to the development of human cancers. P21-Ras mutations have been identified in approximately 30% of human cancers (Bolton et al, Ann. Rep. Med. Chem. (1994) 29: 165-174). Cancer-causing mutations in the P21-Ras protein lead to a constitutively active signaling cascade, causing unregulated activation of the downstream components of the RAF-MEK-ERK-MAP kinase pathway (Magnuson et al., Semin. Cancer Biol. (1994) 5: 247-253). The three RAF kinases which participate in this signaling cascade are known as ARAF, BRAF, and CRAF (Peyssonnaux, C. et al, Biol. Cell (2001) 93: 53-62; Avruch, J., Recent Prog. Horm. Res. (2001) 56: 127-155; Kolch, W., Biochem. J. (2000) 351: 289-305). These RAF kinase isoforms are all activated by Ras, and thus are activated in cancers that result from mutated and upregulated p21-Ras protein activity. In addition to activation of this signaling cascade at the initial p21-Ras protein level, mutations have also been found in BRAF kinase which results in activation of the cascade downstream from p21-Ras (Davies, H., et al, Nature (2002) 417: 949-954). A dominant single site mutation at position 599 in the BRAF kinase was shown to be particularly aggressive and linked to approximately 80% of the observed human malignant melanomas. This mutation substitutes the negatively charged amino acid glutamic acid for the normally occurring neutral amino acid valine. This single site mutation is sufficient to render the mutated BRAF kinase constitutively active, resulting in signaling pathway dysregulation and human cancer. Hence small molecule inhibitors of BRAF kinase are a rational approach to the treatment of human malignancy, whether the signaling mutation is at the level of the upstream p21-Ras protein or at the level of BRAF kinase.
The MAP kinase p38α has recently been identified as an important mechanistic target for the treatment of inflammatory diseases. Inhibition of the MAP kinase p38-alpha has been demonstrated to result in the suppression the production and release the proinflammatory mediators TNF-alpha, IL-1 beta, IL-6, IL-8 and other proinflammatory cytokines (Chen, Z. et al, Chem. Rev. (2001) 101: 2449). Recently, p38-alpha kinase has been implicated in the regulation of tissue factor expression in monocytes, suggesting a role for inhibition of p38-alpha kinase in the treatment of thrombotic disorders and atherosclerosis (Chu, A. J., et al, J. Surg. Res. (2001) 101: 85-90; Eto, M., et al, Circulation (2002) 105: 1756-1759). The p38-alpha kinase has also been shown to be involved in thrombin-induced proinflammatory conditions (V. Marin, et al, Blood, Aug. 1, 2001, 98: 667-673). Validation of this approach has been achieved by the successful application of various protein therapeutic agents for the treatment of severe chronic inflammatory disease. Monoclonal antibodies to TNF have shown effectiveness in the treatment of rheumatoid arthritis, ulcerative colitis, and Crohn's disease (Rankin, E. C. C., et al, British J. Rheum. (1997) 35: 334-342; Stack, W. A., et al, Lancet (1997) 349: 521-524). Enbrel (etanercept), a soluble TNF receptor, has been developed by Immunex, Inc., and marketed currently by Amgen for the treatment of rheumatoid arthritis (Brower et al, Nature Biotechnology (1997) 15: 1240; Pugsley, M. K., Curr. Opin. Invest. Drugs (2001) 2: 1725). Ro 45-2081, a recombinant soluble TNF-alpha receptor chimeric protein, has also shown effectiveness in the treatment of the acute phase of lung injury and in animal models of allergic lung disease (Renzetti, et al, Inflamm Res. (1997) 46: S143). Remicade (infliximab) is a monoclonal TNF-alpha antibody that has shown effectiveness in the treatment of rheumatoid arthritis and Crohn's disease (Bondeson, J. et al, Int. J. Clin. Pract. (2001) 55: 211).
Importantly, small molecule inhibitors of kinase activity have been shown to produce therapeutic benefit as anticipated. The most important example thus far is Gleevec (Imatinib), which is an inhibitor of BCR-Abl kinase (J. Zimmermann et al, WO 99/03854; N. von Bubnoff et al, Cancer Research (2003) 63: 6395; B. J. Druker et al, Nature Medicine (1996) 2: 561; J. Zimmermann et al, Bioorganic and Medicinal Chemistry Letters (1997) 7: 187). Gleevec has been shown to produce clinical remissions in CML patients. However, resistance to the effects of Gleevec have often been encountered (M. E. Gorre et al, Science (2001) 293: 876). Over 17 mutations of Bcr-Abl kinase have been associated with Gleevec resistance (N. von Bubnoff et al, Lancet (2002) 359: 487; S. Branford et al, Blood (2002) 99: 3472; C. Roche-Lestienne et al, Blood (2002) 100: 1014; N. P. Shah et al, Cancer Cell (2002) 2: 117; A. Hochhaus et al, Leukemia (2002) 16: 2190; H. K. Al-Ali et al, Hematology (2004) 5: 55). These mutations are primarily found in the kinase active site domain of Bcr-Abl, and frequently occur in regions proximal to the ATP binding pocket.

The majority of small molecule kinase inhibitors that have been reported have been shown to bind in one of three ways. Most of the reported inhibitors interact with the ATP binding domain of the active site and exert their effects by competing with ATP for occupancy. Other inhibitors have been shown to bind to a separate hydrophobic region of the protein known as the “DFG-in-conformation” pocket, and still others have been shown to bind to both the ATP domain and the “DFG-in-conformation” pocket. Examples specific to inhibitors of RAF kinases can be found in Lowinger et al, Current Pharmaceutical Design (2002) 8: 2269-2278; Dumas, J. et al., Current Opinion in Drug Discovery & Development (2004) 7: 600-616; Dumas, J. et al, WO 2003068223 A1 (2003); Dumas, J., et al, WO 9932455 A1 (1999), and Wan, P. T. C., et al, Cell (2004) 116: 855-867
Physiologically, kinases are regulated by a common activation/deactivation mechanism wherein a specific activation loop sequence of the kinase protein binds into a specific pocket on the same protein which is referred to as the switch control pocket. Such binding occurs when specific amino acid residues of the activation loop are modified for example by phosphorylation, oxidation, or nitrosylation. The binding of the activation loop into the switch pocket results in a conformational change of the protein into its active form (Huse, M. and Kuriyan, J. Cell (109) 275-282.)